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Originally published In Press as doi:10.1074/jbc.M606847200 on December 9, 2006

J. Biol. Chem., Vol. 282, Issue 7, 4908-4915, February 16, 2007
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Archaeal Minichromosome Maintenance (MCM) Helicase Can Unwind DNA Bound by Archaeal Histones and Transcription Factors*

Jae-Ho Shin{ddagger}, Thomas J. Santangelo§1, Yunwei Xie§2, John N. Reeve§, and Zvi Kelman{ddagger}3

From the {ddagger}Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850 and the §Department of Microbiology, Ohio State University, Columbus, Ohio 43210

Received for publication, July 18, 2006 , and in revised form, November 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein-DNA complexes must be disassembled to facilitate DNA replication. Replication forks contain a helicase that unwinds the duplex DNA at the front of the fork. The minichromosome maintenance helicase from the archaeon Methanothermobacter thermautotrophicus required only ATP to unwind DNA bound into complexes by the M. thermautotrophicus archaeal histone HMtA2, transcription repressor TrpY, or into a transcription pre-initiation complex by M. thermautotrophicus TATA-box-binding protein, transcription factor B, and RNA polymerase. In contrast, the minichromosome maintenance helicase was unable to unwind DNA bound by this archaeal RNA polymerase in a stalled transcript-elongating complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA is bound in vivo into complexes by many different proteins, most of which presumably must be displaced to facilitate DNA replication. As DNA helicases are located at the front of the replication machinery, they seem likely to participate in displacing such proteins from DNA, and consistent with this, a number of helicases have been shown to be capable of displacing proteins from DNA. Both Escherichia coli DNA helicase I and Rep protein have the ability to disassociate the LacI repressor from lacO DNA (1), and DnaB can remove Epstein-Barr virus nuclear protein 1 from its binding site on DNA (2). The yeast Pif1 helicase can displace telomerase from telomeric DNA (3), and the yeast Srs2 and bacterial UvrD helicases have been shown to displace Rad51 and RecA, respectively, from single-stranded DNA (ssDNA)4 (4-6). E. coli RecBCD and simian virus 40 large T-antigen helicases have been shown to unwind histone-bound DNA (7). However, in vivo, histone acetylation also aids eukaryotic replication fork progress by destabilizing chromatin and eukaryotic histone-DNA interactions (8). Consistent with the coordination of chromatin destabilization and eukaryotic DNA replication, a human replicative helicase minichromosome maintenance (MCM) protein has been shown to interact with both histones (9) and a histone acetyl transferase (10).

The machineries responsible for DNA replication and transcription in Archaea have fewer components than their eukaryotic counterparts, but the proteins that are present are closely related in sequence and structure to eukaryotic proteins (11, 12). The reduced complexity of the archaeal systems makes them attractive for experimental investigation, both as inherently interesting systems in their own right and as simpler models directly relevant to understanding eukaryotic DNA replication and transcription. With this in mind, we have established robust in vitro DNA- and RNA-synthesizing systems using purified archaeal components that originate from Methano-thermobacter thermautotrophicus and have begun to investigate their regulation (13-15).

M. thermautotrophicus has a single MCM helicase that is thought to function as the replicative helicase. Biochemical studies have established that this enzyme has ATP-dependent 3' -> 5' helicase activity and DNA-dependent ATPase activity, that it can bind and translocate along single- and double-stranded DNA and can unwind DNA-RNA hybrid substrates while translocating along the DNA strand (12). In many Archaea, including M. thermautotrophicus, genomic DNA is bound in vivo by archaeal histones into archaeal nucleosomes, complexes in which ~90 bp of DNA are circularized around an archaeal histone tetramer (12, 16, 17). In structure and DNA constraining properties, archaeal nucleosomes closely resemble eukaryotic tetrasomes, the structure formed at the center of the eukaryotic nucleosome, where DNA is wrapped around eukaryotic (H3+H4)2 histone tetramer (18). It was previously established that the presence of an archaeal histone slowed but did not block transcript elongation by M. thermautotrophicus RNA polymerase (19). The experiments reported here were undertaken first to determine whether movement of the M. thermautotrophicus MCM helicase, the leading component of the archaeal DNA replication machinery, was also sensitive to the presence of an archaeal nucleosome. After establishing that DNA unwinding by the archaeal MCM helicase was not prevented by the presence of an archaeal nucleosome formed by HMtA2, a histone from M. thermautotrophicus, experiments were undertaken to determine whether the archaeal helicase could also unwind the strands of a DNA molecule bound by a M. thermautotrophicus transcription regulator (TrpY), transcription pre-initiation complex, or stalled transcript-elongating complex (14, 15, 19). The results obtained demonstrate that the archaeal MCM helicase requires only ATP to unwind DNA bound by an archaeal histone, TrpY, and transcription pre-initiation complexes but has only minimal ability to disassemble a transcription elongation complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Labeled and unlabeled nucleotides were purchased from GE Healthcare and streptavidin-coated paramagnetic beads from Promega (Madison, WI). Oligonucleotides (see Fig. 1) were synthesized by the DNA facility at the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute (Rockville, MD) or purchased from Integrated DNA Technologies (Coralville, IA).

Purification of M. thermautotrophicus Proteins—The procedures and assays used to purify the M. thermautotrophicus MCM helicase, MCM (K324A) variant, TrpY, HMtA2, TATA-box-binding protein (TBP), transcription factor B (TFB), and RNA polymerase (RNAP), and the properties of these purified proteins have been described previously (14, 15, 19-21).

Preparation of Helicase Substrates—Helicase substrates, with the sequences shown in Fig. 1, were generated by complementary oligonucleotide hybridization and purified as described previously (22). One DNA strand of each substrate was 5' endlabeled (indicated by an asterisk in Fig. 1) by incubation with [{gamma}-32P]ATP and T4 polynucleotide kinase. Complexes were generated by incubation of the appropriate DNA (0.2 pmol) with HMtA2 (5 pmol) or TrpY (20 pmol) in 20 µl of helicase buffer (20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 2 mM dithiothreitol, and 100 µg/ml bovine serum albumin) or with RNAP (1 pmol), TFB (0.2 pmol) and TFB (0.2 pmol) in 20 µl of transcription buffer (20 mM Tris-HCl (pH 8), 10 mM MgCl2, and 120 mM KCl) at 60 °C for 10 min.

MCM Helicase Assays—Reaction mixtures (15 µl) contained 10 fmol of 32P-labeled DNA substrate (~30,000 cpm), 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 2 mM dithiothreitol, 100 µg of bovine serum albumin/ml, 5 mM ATP, and MCM as indicated in the figure legends. Samples were taken after increasing times of incubation at 60 °C and helicase activity terminated by addition of 5 µl of gel loading buffer (100 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue, and 50% glycerol). The products were separated by electrophoresis at 22 °C through non-denaturing gels that contained 4-8% polyacrylamide depending on the DNA substrate. After electrophoresis for 1.5 h at 200 V in 45 mM Tris, 45 mM boric acid, 0.5 mM EDTA (pH 8), the locations and abundance of 32P-labeled molecules were determined by phosphorimaging (GE Healthcare). Helicase activity calculations were based on the amount of single-stranded DNA generated when proteins were not present or by the reduction in a protein-bound substrate.

Assay of Helicase Disruption of Elongation Complexes—Transcription pre-initiation complexes, assembled as described above with 5'-biotinylated DNA substrates, were incubated at 60 °C for 15 min with 200 µM ATP, 200 µM GTP, 10 µM CTP, and 1 µCi [{alpha}-32P]CTP/µl added to allow initiation and transcript elongation to the end of the U-less cassette (at position 25). The resulting complexes were diluted (final concentration of 0.66 nM) in helicase buffer (24 mM Hepes-NaOH (pH 7.5), 24 mM sodium acetate, 7.5 mM magnesium acetate, 1 mM dithiothreitol, 100 µg of bovine serum albumin/ml) and incubated with MCM helicase, with or without 5 mM ATP, at 60 °C for 32 min before being transferred to 23 °C. The M. thermautotrophicus MCM helicase is active at 60 °C but not at 23 °C, whereas the elongation complexes are stable at both temperatures (15). Washed streptavidin-coated paramagnetic beads were added to each sample, and after incubation with gentle agitation for 10 min, the beads were removed to obtain pellet (P) and supernatant (S) fractions. The nucleic acids present in both fractions were purified, dissolved in 95% formamide in TBE (90 mM Tris, 90 mM boric acid, 1 mM EDTA (pH 8)), 0.1% bromphenol blue, 0.1% xylene cyanol, and separated by electrophoresis for 3 h at 1.5 kV through 15% polyacrylamide gels that contained 8 M urea run in TBE (15). The locations and quantities of the 32P-labeled transcripts present were determined by phosphorimaging.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The M. thermautotrophicus MCM helicase was shown previously to have the ability to remove streptavidin molecules from biotinylated DNA (22) and Epstein-Barr virus nuclear protein 1 protein from DNA.5 The experiments described below were undertaken to determine whether this archaeal enzyme could unwind DNA bound by proteins that it would encounter naturally during replication of the M. thermautotrophicus genome.

Archaeal Histone Binding Does Not Inhibit MCM Helicase Activity—The genome of M. thermautotrophicus is bound and compacted by archaeal histones in vivo into archaeal nucleosomes (17). The ability of the MCM helicase to unwind DNA bound by HMtA2, the most abundant archaeal histone in M. thermautotrophicus, was therefore determined using a DNA substrate that contained a 60 bp of sequence (designated Selex1; Fig. 1A) to which HMtA2 binds with high affinity and assembles to form a positioned archaeal nucleosome (19). As shown in Fig. 2A and quantified in Fig. 2B, the MCM helicase generated single-stranded DNA from this substrate equally efficiently when the DNA was protein-free or bound by HMtA2 into an archaeal nucleosome (Fig. 2A, lanes 3-5 and 7-9, respectively). This substrate DNA was not unwound by the MCM (K324A) mutant that lacked helicase activity (Fig. 2A, lane 11) or in reaction mixtures that did not contain ATP (Fig. 2A, lane 10). Time course experiments confirmed that the rate and extent of substrate unwinding were essentially the same, and that unwinding by the MCM helicase occurred with no apparent delay when the DNA was bound by HMtA2 or was supplied as protein-free DNA (Fig. 2, C and D).

TrpY Binding Slows but Does Not Block Helicase Activity—In contrast to HMtA2 that, as a histone, makes contacts primarily with the sugar-phosphate backbone (23) and binds to essentially all DNA sequences, TrpY is a M. thermautotrophicus transcription repressor that binds specifically to TRP-box sequences (consensus TGTACA) that are located upstream of the trpY, trpEGCFBAD, and trpB2 genes in the M. thermautotrophicus genome (14). A helicase substrate was therefore constructed that contained the regulatory region, including the four TRP-box sequences, from upstream of the M. thermautotrophicus trpEGCFBAD operon (Fig. 1B), and the ability of the MCM helicase to unwind this DNA was assayed when protein-free and when bound by TrpY (Fig. 3). TrpY binding did not inhibit DNA unwinding (Fig. 3, A and B) but appeared to reduce the initial rate of unwinding (Fig. 3, C and D). However, after ~15 min at 60 °C, the rate of DNA unwinding was essentially the same with or without TrpY present, consistent with TrpY binding presenting a transient barrier to MCM helicase activity. The TrpY-bound substrate was not unwound by the MCM (K324A) mutant that lacked helicase activity (Fig. 3A, lane 11) or in reaction mixtures that did not contain ATP (Fig. 3A, lane 10).


Figure 1
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  		FIGURE 1.
Helicase substrates used in this study. The DNA sequences of the oligonucleotides used for the study are shown. Shaded boxes indicate protein binding sites, and the labeled strands are marked with asterisks.

 
Archaeal Pre-initiation Transcription Complexes Do Not Block MCM Helicase Activity—Transcription pre-initiation complexes containing the archaeal general transcription factors TBP and TFB and archaeal RNAP assemble spontaneously when incubated with DNA that contains an archaeal promoter and the downstream site of transcription initiation (12). DNA helicase substrates were therefore constructed with the promoter for the hmtB gene from M. thermautotrophicus positioned to direct transcription initiation at the start of a 25 bp of U-less cassette (Fig. 1, C and D) (14, 15). These substrates were incubated with M. thermautotrophicus TBP, TFB, and RNAP to assemble transcription pre-initiation complexes. Pre-initiation complexes spontaneously isomerize under these conditions to yield an open complex, with the two DNA strands separated from approximately -3to +8, relative to the site of initiation (+1), to form a transcription bubble.6 The ability of the MCM helicase to unwind this DNA when bound into pre-initiation complexes (Fig. 4) was determined when the directions of helicase movement and transcription were the same (co-direction; Fig. 4, A-D) and opposite (head-on direction; Fig. 4, E-H). Within a pre-initiation complex, there are specific TFB-DNA, TBP-DNA, and limited RNAP-DNA contacts upstream of the site of transcription initiation, extensive RNAP-template strand DNA contacts within the main channel of RNAP, and extensive RNAP-DNA and limited TFB-DNA contacts downstream of the site of initiation (24, 25). Given this organization, when the direction of helicase unwinding and transcription were the same, helicase would first approach and presumably be required to disrupt TFB-DNA contacts, whereas helicase unwinding in the direction opposite to transcription would presumably first encounter the interactions of RNAP with down-stream DNA. In the absence of transcription factors, the MCM helicase unwound both the co-direction and head-on substrates efficiently (Fig. 4, A and E, lanes 3-6). The rate and extent of unwinding was reduced, but not prevented, by the presence of a pre-initiation complex, oriented in either the same (Fig. 4, A-D) or opposite (Fig. 4, E-H) direction to helicase movement. Neither of the substrates was unwound by the MCM (K324A) mutant that lacked helicase activity (Fig. 4, A and E, lanes 7 and 13).


Figure 2
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  		FIGURE 2.
MCM helicase unwinding of DNA bound by the archaeal histone HMtA2. The reaction assayed is illustrated at the top of the figure with the location of the 32P-label identified by an asterisk. A, result of the helicase assay with archaeal histone-bound substrate by increasing amount of MCM concentration. Reaction mixtures (15 µl) that contained 10 fmol of DNA or 10 fmol of DNA plus HMtA2 at a histone dimer to DNA molar ratio of 12:1 were incubated for 30 min with 5 mM ATP at 60 °C in the presence of 0, 3, 9, or 27 nM MCM helicase (as monomers). The reaction products were separated by non-denaturing gel electrophoresis, and 32P-labeled molecules were visualized and quantified by phosphorimaging. The control lanes contained: 1, boiled substrate; 2, untreated substrate; 6, HMtA2 bound substrate; 10, the products of a reaction mixture incubated with 27 nM MCM (as monomer) without ATP; and 11, the products of a reaction mixture that contained 27 nM of the MCM (K324A) variant that lacked helicase activity. B, the percentage of 32P-labeled single-stranded DNA displaced from the substrate in 30 min in the absence (free, closed circles) or when bound by HMtA2 (histone, open squares). Error bars indicate the standard deviation from three independent experiments. C, result of the helicase assay with archaeal histone bound substrate with increasing length of incubation time. Reaction mixtures (15 µl) that contained 10 fmol of DNA or 10 fmol of DNA plus HMtA2 at a histone dimer to DNA molar ratio of 12:1 were incubated for 2, 4, 8, 16, and 32 min at 60 °C in the presence of 27 nM MCM helicase (as monomer). The control lanes contained: 1, boiled substrate; 2, untreated substrate; 8, HMtA2 bound substrate. D, the percent of 32P-labeled ssDNA displaced from the substrate in the absence (free, closed circles) or presence of HMtA2 (histone, open squares). Error bars indicate the standard deviation from three independent experiments.

 


Figure 3
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  		FIGURE 3.
MCM helicase unwinding of DNA bound by the archaeal repressor TrpY. The reaction assayed is illustrated at the top of the figure with the location of the 32P-label identified by the asterisk. A, result of the helicase assay with TrpY bound substrate by increasing amount of MCM concentration. Reaction mixtures (15 µl) containing 10 fmol of DNA or 10 fmol of DNA plus TrpY at a protein monomer to DNA molar ratio of 100:1 were incubated for 30 min with 5mM ATP at 60 °C in the presence of 0, 3, 9, or 27 nM MCM helicase (as monomer). The reaction products were separated by non-denaturing gel electrophoresis, and 32P-labeled molecules were visualized and quantified by phosphorimaging. The control lanes contained: 1, boiled substrate; 2, untreated substrate; 7, TrpY bound substrate; 11, the products of a reaction mixture incubated with 27 nM MCM (as monomer) without ATP; and 12, the products of a reaction mixture that contained 27 nM MCM (K324A) variant that lacked helicase activity. B, the percent of 32P-labeled ssDNA displaced from the substrate in 30 min in the absence of (free, closed circles) or when bound by TrpY (TrpY, open squares). Error bars indicate the standard deviation from three independent experiments. C, result of the helicase assay with TrpY bound substrate with increasing length of incubation time. Reaction mixtures (15 µl) that contained 10 fmol of DNA or 10 fmol of DNA plus TrpY at a protein monomer to DNA molar ratio of 100:1 were incubated for 2, 4, 8, 16, and 32 min at 60 °C in the presence of 27 nM MCM helicase (as monomer). The control lanes contained: 1, boiled substrate; 2, untreated substrate; 8, TrpY bound substrate. D, the percent of 32P-labeled ssDNA displaced from the substrate in the absence (free, closed circles) or presence of TrpY (TrpY, open squares). Error bars indicate the standard deviation from three independent experiments.

 


Figure 4
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  		FIGURE 4.
MCM helicase unwinding of DNA bound by the archaeal pre-initiation complex. The reactions assayed are illustrated at the top of the figure with the location of the 32P-label identified by the asterisk. Results of helicase assays with transcription machinery bound in the co-direction (Fig. 1C, A-D) and head-on (Fig. 1D, E-H) substrates. Reaction mixtures (15 µl) containing 10 fmol of DNA or 10 fmol of DNA with transcription initiation complexes assembled at the promoter were incubated for 60 min with 5 mM ATP at 60 °C in the presence of 0, 9, 27, 81, or 243 nM MCM helicase (as monomer) (panels A and E) or with increasing length of incubation time (panels C and G). The reaction products were separated by non-denaturing gel electrophoresis, and 32P-labeled molecules were visualized and quantified by phosphorimaging. The control lanes contained: 1, boiled substrate; 2, untreated substrate; 8, substrate with promoter-bound pre-initiation complexes; 7 and 19; the products of reaction mixtures incubated with 243 nM MCM (K324A) variant that lacked helicase activity. The percent of 32P-labeled ssDNA displaced from the substrate or gel-shifted substrate in the absence of transcription factors (free, closed circles) or when bound into pre-initiation complex (pre-initiation, open squares) is shown. Error bars indicate the standard deviation from three independent experiments. B and F, different MCM concentrations; D and H, different incubation times.

 
An Archaeal Transcription Elongation Complex Blocks MCM Helicase Activity—Transcription elongation complexes that contain a nascent transcript, RNAP, and template DNA are very stable in vitro, and if disrupted, do not reassemble. Elongation complexes that contain M. thermautotrophicus RNAP and a nascent transcript stalled because of nucleotide deprivation remain intact for many hours (15). The ability of the MCM helicase to disrupt such complexes was investigated using DNA templates on which the helicase would unwind the DNA in the same (co-direction) and opposite (head-on) direction to RNAP movement. A 5'-biotin moiety was attached to the non-template strand of the co-directional substrate (Fig. 1C) and to the template strand of the head-on substrate (Fig. 1D) to facilitate the removal of the DNA and any DNA-containing complexes from solution by binding to streptavidin-coated paramagnetic beads. Both substrates were rapidly unwound by the MCM helicase in the absence of transcription factors (results not shown) demonstrating that the presence of a 5'-biotin molecule had no detrimental effect on the helicase activity. For these experiments, the substrate DNA was not labeled but was incubated with TFB, TBP, RNAP, ATP, GTP, CTP, and [{alpha}-32P]CTP to obtain elongation complexes in which a stalled 32P-labeled U-less transcript was present. The presence of the RNAP, stalled at the end of the 25 bp of U-less cassette, sterically prevented any additional initiation events. If such elongation complexes were disrupted by exposure to the MCM helicase, the 32P-labeled transcript and RNAP would be released from the DNA and would remain in solution after removal of the DNA by attachment to streptavidin coated beads (I in the scheme on top of Fig. 5). In the absence of UTP, stalled elongation complexes were obtained using both DNA substrates but, whereas most of the complexes formed on the co-directional substrate contained a 25 nt transcript (Fig. 5, lanes 1 and 2), most of the complexes formed on the head-on substrate contained a 21 nt transcript (Fig. 5, lanes 15 and 16). The 21 nt transcript resulted from transcription to 25 followed by RNAP back-tracking and nascent transcript cleavage (data not shown), presumably resulting from an unfavorable interaction of the stalled elongation complex with the downstream forked DNA. In control experiments, the stalled 21 and 25 nt transcripts were extended beyond 25 when UTP was added (data not shown) and were totally removed from solution when the elongation complexes were incubated with paramagnetic beads (Fig. 5, lanes 3, 5, 17, and 19; pellet (P) fractions), consistent with being present in complexes that contained the RNAP and substrate DNA. Incubation with the MCM helicase and ATP had no significant disruptive effect on the elongation complexes formed on either substrate (Fig. 5, lanes 7-12 and 21-26).


Figure 5
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  		FIGURE 5.
MCM helicase unwinding is blocked by a stalled elongation complex. The reaction assayed with the co-directional substrate is illustrated at the top of the figure (the head-on substrate was assayed in the same way). In the illustration, I shows the expected results if the MCM helicase can unwind the elongation complex substrate and release the transcript to solution, whereas II shows the expected results if the helicase cannot unwind the substrates. S, supernatant; P, pellet. 32P-Labeled transcripts elongated from complexes stalled at the end of the U-less cassette in 15 min from the co-direction (Fig. 1C) or head-on (Fig. 1D) substrates were used for the assay. The substrates were incubated for 32 min at 60 °C in the presence of 27, 81, or 243 nM MCM helicase (as monomer) as described under "Materials and Methods." Reaction mixtures were then transferred to 23 °C and incubated with streptavidin-coated paramagnetic beads for 10 min followed by separation into pellet (P) and supernatant (S) fractions. Transcripts were separated by denaturing gel electrophoresis, and 32P-labeled molecules were visualized and quantified by phosphorimaging. Control lanes are 1, 15 and 2, 16, which are the elongation complexes formed after 5 and 15 min, respectively; lanes 3-6 and 17-19 contained no MCM; lanes 13, 14, 27, and 28 contained no ATP; and lanes 1-4 and 15-18 were not incubated at 60 °C.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The MCM helicase from the archaeon M. thermautotrophicus has the ability to unwind DNA bound by a variety of different M. thermautotrophicus proteins when provided with ATP. Most notably, HMtA2 histone wrapping of DNA into an archaeal nucleosome had virtually no detrimental effect on the extent or rate at which the MCM unwound the histone-bound DNA (Fig. 2). This seems particularly significant given that archaeal histones are sufficiently abundant in M. thermautotrophicus to package most, if not all, of the 1.7 Mbp genome into archaeal nucleosomes (17). The helical structure of double-stranded DNA is essential for histone-DNA interaction (23), and as the advancing helicase generates single-stranded DNA, these interactions will be lost resulting in histone-DNA complex disassembly and allowing unimpeded MCM progress. DNA unwinding by eukaryotic helicases presumably also generates single-stranded DNA that destabilizes eukaryotic histone-DNA binding, but whereas archaeal histones bind DNA only through histone fold contacts, eukaryotic histones also bind DNA through histone tail residues. It seems likely that histone tail acetylation first reduces the affinity of the eukaryotic histone tail regions for DNA (8) before the approach of the eukaryotic helicase disassembles the nucleosome core. Intriguingly, the human MCM helicase is associated with a histone acetyltransferase (9), and acetylation of histone tail lysine residues is thought to contribute substantially to reducing histone-DNA affinity (8).

DNA unwinding by the archaeal MCM helicase was not blocked by the TrpY repressor or by a pre-initiation complex, although the presence of these transcription factors on the substrate DNA did marginally reduce the rate and extent of unwinding. In contrast, in >90% of cases, the presence of a stalled transcription elongation complex blocked helicase unwinding of the DNA. This is consistent with the report that the bacteriophage T4 replication machinery paused transiently in head-on collisions with a transcription complex, although pausing was not observed when the collision was co-directional (26). Head-on collisions of the E. coli DNA replication and transcription machineries have been reported to reduce replication fork movement in vivo (27, 28). All seven rRNA operons in the E. coli genome are oriented such that transcription and replication occur in the same direction, presumably to facilitate disassembly of transcription complexes during replication. Studies with Saccharomyces cerevisiae and Xenopus laevis have similarly shown that the eukaryotic DNA replication machinery transiently stalls in vivo in head-on but not in co-directional collisions with RNAP (29, 30). These results argue that a full replication fork, rather than an isolated helicase, may be required to disrupt transcription elongation complexes and/or that additional cellular factors may exist that facilitate removal of elongation complexes to allow passage of the replication fork. Most bacterial and eukaryotic transcription termination factors are known to follow and attack from upstream of an elongating complex, consistent with disruption being most effective in co-directional interactions. The results reported demonstrate that the M. thermautotrophicus MCM alone does have inherent ability to unwind protein-DNA complexes, but it is almost certain that in vivo this enzyme is part of a much larger replication complex (31) that has the added ability to displace transcription factors and RNAP from DNA. Helicase activities have, in fact, been shown to be stimulated by the presence of DNA polymerases (32, 33), and the observed interactions of MCM with RNAP in eukaryotes (34, 35) may also aid helicase unwinding of RNAP-bound DNA. It seems most likely that the ability of the archaeal helicase to unwind DNA bound by an archaeal histone, TrpY, and the transcription initiation complex and its inability to unwind the DNA in an elongation complex reflects the differences in the stabilities of these protein-DNA complexes. DNA binding by TrpY, archaeal histones, and the initiation components is inherently dynamic, and their transient dissociations from the DNA substrate may provide the helicase with access and adequate opportunity to unwind the DNA. In contrast, elongation complexes are extremely stable with the RNAP bound to the DNA through many contacts and a tightly packed RNA-DNA hybrid positioned and bound within the RNAP active site (36).

All archaeal MCM helicases putatively identified to date have very similar sequences, and those investigated directly have very similar activities, processivities, and DNA binding abilities. It seems likely that all of these archaeal enzymes will have the ATP-dependent inherent ability to unwind archaeal histone- and transcription factor-bound DNA established here for the M. thermautotrophicus MCM helicase. However, not all Archaea have histones, and some have both archaeal histones and one or more members of several non-histone archaeal chromatin protein families such as HTa, Sul7d, Sso10a, Sso10b (Alba), and MC1 (12). The helicases in these Archaea must therefore also have the ability to unwind DNA bound into the variety of different protein-DNA complexes formed by these non-histone genome-compacting proteins.


   FOOTNOTES
 
* This work was supported in part by Research Scholar Grant RSG-04-050-01-GMC from the American Cancer Society (to Z. K.) for research at the Center for Advanced Research in Biotechnology and by Grants DE-FG02-87ER13731 and GM53185 (to J. N. R.) from the Department of Energy and the National Institutes of Health, respectively, for research at the Ohio State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by National Institutes of Health postdoctoral fellowship (GM073336-01). Back

2 Present address: Lombardi Cancer Center, Georgetown University, Washington, D. C. 20057. Back

3 To whom correspondence should be addressed: Univ. of Maryland Biotechnology Inst., Center for Advanced Research in Biotechnology, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 240-314-6294; Fax: 240-314-6245; E-mail: kelman{at}umbi.umd.edu.

4 The abbreviations used are: ssDNA, single-stranded DNA; MCM, minichromosome maintenance; RNAP, RNA polymerase; TBP, TATA-box-binding protein; TFB, archaeal transcription factor B; TBE, Tris borate-EDTA; nt, nucleotide. Back

5 J.-H. Shin and Z. Kelman, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Kathleen Sandman for providing the HMtA2 expression plasmid.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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